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This strain is recommended by ATCC for use in the tests described in ASTM Standard Test Method E1366-91 where only the taxon is specified.

Production in deuterium oxide

Biosafety Level

1

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

Isolation

freshwater, (?)

Product Format

freeze-dried

Type Strain

no

Comments

This strain is recommended by ATCC for use in the tests described in ASTM Standard Test Method E1366-91 where only the taxon is specified.

3. While cells are centrifuging prepare a 10% (v/v) solution of sterile methanol in fresh medium.

4. Mix the cell preparation and the 10% methanol in equal portions. Thus, the final concentration will be 106 - 107 cells/ml and 5% (v/v) Methanol. The time from the mixing of the cell preparation and methanol stock solution to the beginning of the freezing process should be no less than 5 min and no greater than 15 min.

6. Place the vials in a controlled rate freezing unit. From room temperature cool at -1°C/min to -40°C. If the freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through the heat of fusion. At -40°C plunge into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing apparatus. Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen. (The cooling rate in this apparatus is approximately

-1°C/min.)

7. The frozen preparations should be stored in either the vapor or liquid phase of a nitrogen refrigerator. Frozen preparations stored below -130°C are stabile indefinitely. Those stored at temperatures above -130°C are progressively less stabile as the storage temperature is elevated. Vials should not be stored above -55°C.

8. To establish a culture from the frozen state place an ampule in a water bath set at 35°C. Immerse the vial just to a level just above the surface of the frozen material. Do not agitate the vial.

9. Immediately after thawing, do not leave in the water bath, aseptically remove the contents of the ampule and add to a centrifuge tube containing 5 ml of ATCC medium 5 without agar. Centrifuge at 300 x g for 5 min.

Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.